U.S. patent application number 13/610967 was filed with the patent office on 2013-01-03 for anti-francisella agents.
This patent application is currently assigned to THE OHIO STATE UNIVERSITY RESEARCH FOUNDATION. Invention is credited to Ching-Shih Chen, Hao-Chieh Chiu, John S. Gunn, Samuel Kulp, Larry S. Schlesinger.
Application Number | 20130005788 13/610967 |
Document ID | / |
Family ID | 42352707 |
Filed Date | 2013-01-03 |
United States Patent
Application |
20130005788 |
Kind Code |
A1 |
Chen; Ching-Shih ; et
al. |
January 3, 2013 |
ANTI-FRANCISELLA AGENTS
Abstract
A series of celecoxib derivatives defined by Formula I:
##STR00001## were prepared and evaluated for their ability to
inhibit the gram-negative bacteria Francisella tularensis.
Pharmaceutical compositions including celecoxib derivatives and
their use in methods for treating or preventing infection by
Francisella tularensis in a subject are described.
Inventors: |
Chen; Ching-Shih; (Upper
Arlington, OH) ; Chiu; Hao-Chieh; (Columbus, OH)
; Kulp; Samuel; (Hilliard, OH) ; Gunn; John
S.; (Powell, OH) ; Schlesinger; Larry S.;
(Powell, OH) |
Assignee: |
THE OHIO STATE UNIVERSITY RESEARCH
FOUNDATION
Columbus
OH
|
Family ID: |
42352707 |
Appl. No.: |
13/610967 |
Filed: |
September 12, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12428035 |
Apr 22, 2009 |
|
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13610967 |
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Current U.S.
Class: |
514/406 |
Current CPC
Class: |
A61P 31/04 20180101;
A61P 31/00 20180101; C07D 231/12 20130101 |
Class at
Publication: |
514/406 |
International
Class: |
A61K 31/415 20060101
A61K031/415; A61P 31/04 20060101 A61P031/04 |
Goverment Interests
GOVERNMENT FUNDING
[0001] The present invention was made with government support under
award No. 1-U54-AI-057153, awarded by the NIH/NIAID Regional Center
of Excellence for Bio-defense and Emerging Infectious Diseases
Research Program. The Government may have certain rights in this
invention.
Claims
1. A method of treating or preventing infection by Francisella
tularensis in a subject, comprising administering a therapeutically
effective amount of a composition including a compound of Formula
I: ##STR00012## wherein: R.sup.1 is selected from the group
consisting of hydrogen, amino, amido, sulfonamidyl, methylsulfinyl,
and ethylsulfinyl moieties, and R.sup.2 is an aryl group, and
wherein R.sup.2 is optionally substituted at substitutable
positions with one of more moieties independently selected from
halo, lower alkyl, lower haloalkyl, lower hydroxyalkyl, hydroxyl,
nitro, amino, carboxyl, and cyano; or a pharmaceutically acceptable
salt thereof.
2. The method of claim 1, wherein the R.sup.2 aryl group is
selected from the group consisting of phenyl, naphthyl, biphenyl,
anthracenyl, and phenanthracenyl aryl groups.
3. The method of claim 2, wherein the optional substituting
moieties for R.sup.2 are independently selected from bromo, chloro,
fluoro, methoxy, trifluoromethyl, methyl, ethyl, propyl, and
butyl.
4. The method of claim 1, wherein the compound is selected from the
group consisting of: Celecoxib;
4-(5-biphenyl-4-yl-3-trifluoromethyl-pyrazol-1-yl)-benzamide;
4-[5-(4'-chloro-biphenyl-4-yl)-3-trifluoromethyl-pyrazol-1-yl]-benzamide;
4-[5-(3',5'-dichloro-biphenyl-4-yl)-3-trifluoromethyl-pyrazol-1-yl]-benza-
mide;
4-[5-(4'-methyl-biphenyl-4-yl)-3-trifluoromethyl-pyrazol-1-yl]-benza-
mide;
4-[3-trifluoromethyl-5-(4'-trifluoromethyl-biphenyl-4-yl)-pyrazol-1--
yl]-benzamide;
4-[5-(3',5'-dimethyl-biphenyl-4-yl)-3-trifluoromethyl-pyrazol-1-yl]-benza-
mide;
4-[5-(4'-tert-Butyl-biphenyl-4-yl)-3-trifluoromethyl-pyrazol-1-yl]-b-
enzamide;
4-[5-(4'-butyl-biphenyl-4-yl)-3-trifluoromethyl-pyrazol-1-yl]-be-
nzamide;
4-(5-anthracen-9-yl-3-trifluoromethyl-pyrazol-1-yl)-benzamide;
4-[5-(4-butyl-phenyl)-3-trifluoromethyl-pyrazol-1-yl]-benzamide;
4-[5-(4'-chloro-biphenyl-4-yl)-3-trifluoromethyl-pyrazol-1-yl]-benzenesul-
fonamide;
4-[5-(3',4',5'-trichloro-biphenyl-4-yl)-3-trifluoromethyl-pyrazo-
l-1-yl]-benzenesulfonamide;
4-(5-naphthalen-1-yl-3-trifluoromethyl-pyrazol-1-yl)-benzenesulfonamide;
5-(4-fluoro-phenyl)-1-(4-ethanesulfonyl-phenyl)-3-trifluoromethyl-1H-pyra-
zole;
1-(4-methanesulfonyl-phenyl)-5-(4'-methyl-biphenyl-4-yl)-3-trifluoro-
methyl-1H-pyrazole;
4-(5-biphenyl-4-yl-3-trifluoromethyl-pyrazol-1-yl)-phenylamine;
4-[5-(3',4',5'-trichloro-biphenyl-4-yl)-3-trifluoromethyl-pyrazol-1-yl]-p-
henylamine;
4-(5-naphthalen-1-yl-3-trifluoromethyl-pyrazol-1-yl)-phenylamine;
4-[5-(6-methoxy-naphthalen-2-yl)-3-trifluoromethyl-pyrazol-1-yl]-phenylam-
ine;
4-[5-(4'-bromo-biphenyl-4-yl)-3-trifluoromethyl-pyrazolyl]-phenylamin-
e; and 5-phenanthren-3-yl-1-phenyl-3-trifluoromethyl-1H-pyrazole;
and their pharmaceutically acceptable salts.
5. The method of claim 1, wherein the compound is selected from the
group consisting of: Celecoxib;
4-[5-(4'-chloro-biphenyl-4-yl)-3-trifluoromethyl-pyrazol-1-yl]-benzamide;
4-[5-(4'-chloro-biphenyl-4-yl)-3-trifluoromethyl-pyrazol-1-yl]-benzenesul-
fonamide;
4-[5-(3',4',5'-trichloro-biphenyl-4-yl)-3-trifluoromethyl-pyrazo-
l-1-yl]-benzenesulfonamide;
4-(5-biphenyl-4-yl-3-trifluoromethyl-pyrazol-1-yl)-phenylamine; and
4-[5-(4'-bromo-biphenyl-4-yl)-3-trifluoromethyl-pyrazolyl]-phenylamine;
and their pharmaceutically acceptable salts.
6. The method of claim 1, wherein the compound is
4-[5-(4'-bromo-biphenyl-4-yl)-3-trifluoromethyl-pyrazolyl]-phenylamine
or a pharmaceutically acceptable salt thereof.
7. The method of claim 1, wherein the Francisella tularensis
infection is inhibited in macrophage cells without significant
toxicity to the macrophage cells.
8. The method of claim 1, wherein the Francisella tularensis is
antibiotic resistant.
9. The method of claim 1, wherein the subject is a human.
10-21. (canceled)
Description
BACKGROUND
[0002] Francisella tularensis is a gram-negative, facultative,
highly virulent bacterium, which causes the zoonotic disease
tularemia. Infection can occur through several routes, but
pneumonic tularemia is the most severe clinical form, with a
mortality rate up to 60 percent in the absence of treatment. F.
tularensis can invade a range of host cells, but its primary target
in vivo is the macrophage. Sjostedt, A, Curr. Opin. Microbiol. 6,
p. 66-71 (2003). After being phagocytosed by macrophages, this
intracellular pathogen can block the fusion of
Francisella-containing phagosomes with lysosomes and escape from
the phagosome into the cytosol where it multiplies. Following
proliferation within macrophages, F. tularensis induces host cell
apoptosis or pyroptosis leading to the release of bacteria and
subsequent infection of new cells.
[0003] Because of the ease with which aerosolized organisms could
potentially be deliberately disseminated, inflicting substantial
morbidity and mortality on large numbers of people, F. tularensis
has been recognized as a potential biological warfare agent and,
consequently, has been classified as a Category A bioterrorism
agent by the U.S. Centers for Disease Control and Prevention.
Unfortunately, the current live attenuated vaccine derived from a
type B strain of F. tularensis has serious drawbacks and is of
limited utility in the face of a bioterror threat. Oyston et al.,
Nat. Rev. Microbiol. 2, p. 967-78 (2004). Moreover, it is believed
that antibiotic-resistant strains of F. tularensis were created in
the early 1990s as biological weapons. Dennis et al., JAMA, 285, p.
2763-73 (2001). Consequently, the development of novel,
antibacterial agents against F. tularensis has become an important
priority.
SUMMARY OF THE INVENTION
[0004] The present invention provides a number of celecoxib
derivatives that demonstrate antibacterial activity against F.
tularensis. Accordingly, one aspect of the invention provides a
method of treating or preventing infection by Francisella
tularensis in a subject, comprising administering a therapeutically
effective amount of a composition including a celecoxib derivative,
or a pharmaceutically acceptable salt thereof, as further defined
herein. In some embodiments, the Francisella tularensis infection
is inhibited in macrophage cells without significant toxicity to
the macrophage cells, and in some embodiments the Francisella
tularensis is antibiotic resistant. In further embodiments, the
subject is a human.
[0005] Another aspect of the invention provides compounds according
to formula I
##STR00002##
[0006] In this aspect of the invention, R.sup.1 of Formula I is
selected from the group consisting of hydrogen, amino, amido,
methylsulfinyl, and ethylsulfinyl moieties, and R.sup.2 is an aryl
group. Group R.sup.2 of formula I is optionally substituted at
substitutable positions with one of more moieties independently
selected from halo, lower alkyl, lower haloalkyl, lower
hydroxyalkyl, hydroxyl, nitro, amino, carboxyl, and cyano. This
aspect of the invention also includes pharmaceutically acceptable
salts of the compounds of formula (I).
[0007] Another aspect of the invention provides pharmaceutical
compositions including a compound of formula I:
##STR00003##
[0008] In this aspect of the invention, R.sup.1 is selected from
the group consisting of hydrogen, amino, amido, methylsulfinyl, and
ethylsulfinyl moieties, and R.sup.2 is an aryl group. Group R.sup.2
of Formula I is optionally substituted at substitutable positions
with one of more moieties independently selected from halo, lower
alkyl, lower haloalkyl, lower hydroxyalkyl, hydroxyl, nitro, amino,
carboxyl, and cyano. The compound of formula I, or a
pharmaceutically acceptable salt thereof; is an active ingredient
in the pharmaceutical composition, and is provided in combination
with a pharmaceutically acceptable liquid or solid carrier or
carriers.
BRIEF DESCRIPTION OF THE FIGURES
[0009] FIG. 1 shows the inhibition of F. tularensis growth by
celecoxib in broth culture, with section (A) showing the structures
of COX-2-specific inhibitors celecoxib (left) and rofecoxib
(right), and section (B) providing a bar graph showing the results
of a bacteria viability assay. F. novicida and LVS were cultured in
modified TSB containing different doses of celecoxib or rofecoxib
at 37.degree. C. Viable F. novicida and LVS were measured as CFU
after 24 or 48 h of incubation, respectively. Columns, mean; bars,
.+-.SD (n=3). Section (C) provides a table showing the results of
the minimum inhibitory concentration (MIC) assay. Each test group
was treated in triplicate and values shown are the results from one
of three independent experiments.
[0010] FIG. 2 provides a table showing the structures and
characteristics of celecoxib and compounds 1-21. The general
structure of these molecules is shown at the top. Each test group
was treated in triplicate and values shown are the results from one
of three independent experiments.
[0011] FIG. 3 shows the effect of celecoxib and its derivatives on
RAW264.7 murine macrophage cells, with section (A) providing a
graph showing the results of a cytotoxicity assay of selected
agents, where RAW264.7 cells were treated with different doses of
celecoxib and compounds (2, 11, 12, 16 and 20) in DMEM supplemented
with 5% FBS for 8 h. The viability of drug-treated cells was
measured using the MTT cell viability assay and expressed as a
percentage of vehicle control (DMSO)-treated cells. Points, mean;
bars, .+-.SD (n=3), and section (B) providing a table showing
comparison of cytotoxicity and antimicrobial efficacy of selected
agents.
[0012] FIG. 4 shows the inhibition of intracellular F. tularensis
by compound 20, with section (A) providing a bar graph showing that
compound 20 decreased the survival of intracellular F. novicida in
RAW264.7 and THP-1 cells, and section (B) showing the decrease of
intracellular survival of F. tualrensis (type A, Schu S4) in THP-1
cells by compound 20. Data are presented as the % survival relative
to vehicle control (DMSO)-treated cells. Columns, mean; bars,
.+-.SD (n=3). *P<0.05.
DETAILED DESCRIPTION OF THE INVENTION
[0013] The inventors have demonstrated that the cyclooxygenase-2
(COX-2)-specific inhibitor celecoxib
(4-[5-(4-methylphenyl)-3-(trifluoromethyl)pyrazol-1-yl]benzenesulfonamide-
) exhibits antibacterial activity against a virulent type A strain
of F. tularensis (Schu S4), the live vaccine strain (LVS) of F.
tularensis (type B strain) and F. tularensis subspecies novicida
(an avirulent subspecies) directly in growth medium. This bacterial
killing, however, was not noted with another COX-2 specific
inhibitor, rofecoxib, despite its higher potency relative to
celecoxib in COX-2 inhibition. Tacconelli et al., Curr. Med. Res.
Opin. 18, p. 503-11 (2002). From a drug discovery perspective, the
unique ability of celecoxib to inhibit the proliferation of F.
tularensis can be pharmacologically exploited as a molecular
platform to develop novel anti-Francisella agents.
Definitions
[0014] The terminology as set forth herein is for description of
the embodiments only and should not be construed as limiting of the
invention as a whole. Unless otherwise specified, "a," "an," "the,"
and "at least one" are used interchangeably. Furthermore, as used
in the description of the invention and the appended claims, the
singular forms "a", "an", and "the" are inclusive of their plural
forms, unless contraindicated by the context surrounding such.
[0015] The terms "comprising" and variations thereof do not have a
limiting meaning where these terms appear in the description and
claims.
[0016] As used herein, the term "organic group" is used for the
purpose of this invention to mean a hydrocarbon group that is
classified as an aliphatic group, cyclic group, or combination of
aliphatic and cyclic groups (e.g., alkaryl and aralkyl groups). In
the context of the present invention, suitable organic groups for
celecoxib derivatives are those that do not interfere with the
celecoxib derivative's anti-francisella activity. In the context of
the present invention, the term "aliphatic group" means a saturated
or unsaturated linear or branched hydrocarbon group. This term is
used to encompass alkyl, alkenyl, and alkynyl groups, for
example.
[0017] As used herein, the terms "alkyl", "alkenyl", and the prefix
"alk-" are inclusive of straight chain groups and branched chain
groups and cyclic groups, e.g., cycloalkyl and cycloalkenyl. Unless
otherwise specified, these groups contain from 1 to 20 carbon
atoms, with alkenyl groups containing from 2 to 20 carbon atoms. In
some embodiments, these groups have a total of at most 10 carbon
atoms, at most 8 carbon atoms, at most 6 carbon atoms, or at most 4
carbon atoms. Lower alkyl groups are those including at most 6
carbon atoms. Examples of alkyl groups include haloalkyl groups and
hydroxyalkyl groups. Cyclic groups can be monocyclic or polycyclic
and preferably have from 3 to 10 ring carbon atoms.
[0018] Unless otherwise specified, "alkylene" and "alkenylene" are
the divalent forms of the "alkyl" and "alkenyl" groups defined
above. The terms, "alkylenyl" and "alkenylenyl" are used when
"alkylene" and "alkenylene", respectively, are substituted. For
example, an arylalkylenyl group comprises an alkylene moiety to
which an aryl group is attached.
[0019] The term "haloalkyl" is inclusive of groups that are
substituted by one or more halogen atoms, including perfluorinated
groups. This is also true of other groups that include the prefix
"halo-". Examples of suitable haloalkyl groups are chloromethyl,
trifluoromethyl, and the like. A halo moiety can be chlorine,
bromine, fluorine, or iodine.
[0020] The term "aryl" as used herein includes carbocyclic aromatic
rings or ring systems. Examples of aryl groups include phenyl,
naphthyl, biphenyl, anthracenyl, phenanthracenyl, fluorenyl and
indenyl. Aryl groups may be substituted or unsubstituted.
[0021] Unless otherwise indicated, the term "heteroatom" refers to
the atoms O, S, or N.
[0022] The term "heteroaryl" includes aromatic rings or ring
systems that contain at least one ring heteroatom (e.g., O, S, N).
In some embodiments, the term "heteroaryl" includes a ring or ring
system that contains 2 to 12 carbon atoms, 1 to 3 rings, 1 to 4
heteroatoms, and O, S, and/or N as the heteroatoms. Suitable
heteroaryl groups include furyl, thienyl, pyridyl, quinolinyl,
isoquinolinyl, indolyl, isoindolyl, triazolyl, pyrrolyl,
tetrazolyl, imidazolyl, pyrazolyl, oxazolyl, thiazolyl,
benzofuranyl, benzothiophenyl, carbazolyl, benzoxazolyl,
pyrimidinyl, benzimidazolyl, quinoxalinyl, benzothiazolyl,
naphthyridinyl, isoxazolyl, isothiazolyl, purinyl, quinazolinyl,
pyrazinyl, 1-oxidopyridyl, pyridazinyl, triazinyl, tetrazinyl,
oxadiazolyl, thiadiazolyl, and so on.
[0023] The terms "arylene" and "heteroarylene" are the divalent
forms of the "aryl" and "heteroaryl" groups defined above. The
terms "arylenyl" and "heteroarylenyl" are used when "arylene" and
"heteroarylene", respectively, are substituted. For example, an
alkylarylenyl group comprises an arylene moiety to which an alkyl
group is attached.
[0024] When a group is present more than once in any formula or
scheme described herein, each group (or substituent) is
independently selected, whether explicitly stated or not. For
example, for the formula --C(O)--NR.sub.2 each R group is
independently selected.
[0025] As a means of simplifying the discussion and the recitation
of certain terminology used throughout this application, the terms
"group" and "moiety" are used to differentiate between chemical
species that allow for substitution or that may be substituted and
those that do not so allow for substitution or may not be so
substituted. Thus, when the term "group" is used to describe a
chemical substituent, the described chemical material includes the
unsubstituted group and that group with nonperoxidic O, N, S, Si,
or F atoms, for example, in the chain as well as carbonyl groups or
other conventional substituents. Where the term "moiety" is used to
describe a chemical compound or substituent, only an unsubstituted
chemical material is intended to be included. For example, the
phrase "alkyl group" is intended to include not only pure open
chain saturated hydrocarbon alkyl substituents, such as methyl,
ethyl, propyl, tert-butyl, and the like, but also alkyl
substituents bearing further substituents known in the art, such as
hydroxy, alkoxy, alkylsulfonyl, halogen atoms, cyano, nitro, amino,
carboxyl, etc. Thus, "alkyl group" includes ether groups,
haloalkyls, nitroalkyls, carboxyalkyls, hydroxyalkyls, sulfoalkyls,
etc. On the other hand, the phrase "alkyl moiety" is limited to the
inclusion of only pure open chain saturated hydrocarbon alkyl
substituents, such as methyl, ethyl, propyl, tert-butyl, and the
like.
[0026] "Treat", "treating", and "treatment", etc., as used herein,
refer to any action providing a benefit to a patient at risk for or
afflicted with a disease, including improvement in the condition
through lessening or suppression of at least one symptom, delay in
progression of the disease, prevention or delay in the onset of the
disease, etc.
[0027] Macrophage cells, as used herein, refers to immune cells of
the innate immune system, and include macrophages, macrophage-like
cells, and macrophage precursors such as monocytes. Macrophage-like
cells include tingible body macrophages, dendritic cells, foam
cells, and multinucleated giant cells.
[0028] Antibiotics, as defined herein, are bactericidal or
bacteriostatic compounds already known in the art. Examples of
known antibiotics include agents that target the bacterial cell
wall, such as penicillins, cephalosporins, agents that target the
cell membranesuch as polymixins, agents that interfere with
essential bacterial enzymes, such as quinolones and sulfonamides,
and agents that that target protein synthesis such as the
aminoglycosides, macrolides and tetracyclines. Additional known
antibiotics include cyclic lipopeptides, glycylcyclines, and
oxazolidinones.
[0029] "Pharmaceutically acceptable" as used herein means that the
compound or composition is suitable for administration to a subject
to achieve the treatments described herein, without unduly
deleterious side effects in light of the severity of the disease
and necessity of the treatment.
[0030] "Inhibit" as used herein refers to the partial or complete
elimination of a potential effect, while inhibitors are compounds
that have the ability to inhibit.
Celecoxib Derivatives
[0031] Celecoxib derivatives, as defined herein, include the
compounds of formula I:
##STR00004##
[0032] Formula 1 provides 3-trifluoromethyl pyrazolyl 1-phenyl core
to which is attached an organic group R.sup.1 at the 4' position of
the phenyl group and an organic group R.sup.2 at the 5' position of
the pyrazolyl group. In celecoxib derivatives, which as defined
herein includes celecoxib itself, the organic group R.sup.1 is
selected from the group consisting of hydrogen, amino, amido,
methylsulfinyl, and ethylsulfinyl moieties, and the organic group
R.sup.2 is an aryl group. The aryl group R.sup.2 is optionally
substituted at substitutable positions with one of more moieties
independently selected from halo, lower alkyl, lower haloalkyl,
lower hydroxyalkyl, hydroxyl, nitro, amino, carboxyl, and
cyano.
[0033] In embodiments of the invention, the R.sup.2 aryl group is
selected from the group consisting of phenyl, naphthyl, biphenyl,
anthracenyl, and phenanthracenyl aryl groups. In further
embodiments, the optional substituting moieties for R.sup.2 are
independently selected from bromo, chloro, fluoro, methoxy,
trifluoromethyl, methyl, ethyl, propyl, and butyl.
[0034] Examples of celecoxib derivatives that can be used for the
treatment of Francisella tularensis infection include celecoxib, as
well as the non-celexoxib derivatives
4-(5-biphenyl-4-yl-3-trifluoromethyl-pyrazol-1-yl)-benzamide
(Compound 1);
4-[5-(4'-chloro-biphenyl-4-yl)-3-trifluoromethyl-pyrazol-1-yl]-benzam-
ide (Compound 2);
4-[5-(3',5-dichloro-biphenyl-4-yl)-3-trifluoromethyl-pyrazol-1-yl]-benzam-
ide (Compound 3);
4-[5-(4'-methyl-biphenyl-4-yl)-3-trifluoromethyl-pyrazol-1-yl]-benzamide
(Compound 4);
4-[3-trifluoromethyl-5-(4'-trifluoromethyl-biphenyl-4-yl)-pyrazol-1-yl]-b-
enzamide (Compound 5);
[0035]
4-[5-(3',5-dimethyl-biphenyl-4-yl)-3-trifluoromethyl-pyrazol-1-yl]--
benzamide (Compound 6);
4-[5-(4'-tert-butyl-biphenyl-4-yl)-3-trifluoromethyl-pyrazol-1-yl]-benzam-
ide (Compound 7);
4-[5-(4'-butyl-biphenyl-4-yl)-3-trifluoromethyl-pyrazol-1-yl]-benzamide
(Compound 8);
4-(5-anthracen-9-yl-3-trifluoromethyl-pyrazol-1-yl)-benzamide
(Compound 9); and
4-[5-(4-butyl-phenyl)-3-trifluoromethyl-pyrazol-1-yl]-benzamide
(Compound 10).
[0036] Celecoxib derivatives further include
4-[5-(4'-chloro-biphenyl-4-yl)-3-trifluoromethyl-pyrazol-1-yl]-benzenesul-
fonamide (Compound 11);
4-[5-(3',4',5'-trichloro-biphenyl-4-yl)-3-trifluoromethyl-pyrazol-1-yl]-b-
enzenesulfonamide (Compound 12);
4-(5-naphthalen-1-yl-3-trifluoromethyl-pyrazol-1-yl)-benzenesulfonamide
(Compound 13);
5-(4-fluoro-phenyl)-1-(4-ethanesulfonyl-phenyl)-3-trifluoromethyl-1H-pyra-
zole (Compound 14);
1-(4-methanesulfonyl-phenyl)-5-(4'-methyl-biphenyl-4-yl)-3-trifluoromethy-
l-1H-pyrazole (Compound 15);
4-(5-biphenyl-4-yl-3-trifluoromethyl-pyrazol-1-yl)-phenylamine
(Compound 16);
4-[5-(3',4',5-trichloro-biphenyl-4-yl)-3-trifluoromethyl-pyrazol-1-y-
l]-phenylamine (Compound 17);
4-(5-naphthalen-1-yl-3-trifluoromethyl-pyrazol-1-yl)-phenylamine
(Compound 18);
4-[5-(6-methoxy-naphthalen-2-yl)-3-trifluoromethyl-pyrazol-1-yl]-phenylam-
ine (Compound 19);
4-[5-(4'-bromo-biphenyl-4-yl)-3-trifluoromethyl-pyrazolyl]-phenylamine
(Compound 20); and
5-phenanthren-3-yl-1-phenyl-3-trifluoromethyl-1H-pyrazole (Compound
21).
[0037] In additional embodiments of the invention, celecoxib
derivatives that exhibit higher activity (e.g., they exhibit lower
minimum inhibitory concentration against Francisella tularensis)
may be preferred. For example, some embodiments include the
compounds celecoxib,
4-[5-(4'-chloro-biphenyl-4-yl)-3-trifluoromethyl-pyrazol-1-yl]-benzamide
(Compound 2);
4-[5-(4'-chloro-biphenyl-4-yl)-3-trifluoromethyl-pyrazol-1-yl]-benzenesul-
fonamide (Compound 11);
4-[5-(3',4',5'-trichloro-biphenyl-4-yl)-3-trifluoromethyl-pyrazol-1-yl]-b-
enzenesulfonamide (Compound 12);
4-(5-biphenyl-4-yl-3-trifluoromethyl-pyrazol-1-yl)-phenylamine
(Compound 16); and
4-[5-(4'-bromo-biphenyl-4-yl)-3-trifluoromethyl-pyrazolyl]-pheny-
lamine (Compound 20). In an additional embodiment, the compound is
4-[5-(4'-bromo-biphenyl-4-yl)-3-trifluoromethyl-pyrazolyl]-phenylamine
(Compound 20) is preferred. Additional embodiments of the use of
celecoxib derivatives for treating infection with Francisella
tularensis also include the pharmaceutically acceptable salts of
these compounds.
[0038] While the compound celecoxib is already known for use as an
antiarthritic agent, and a number of substituted pyrazolyl
benzenesulfonamides have been prepared for treating inflammation,
as described in U.S. Pat. No. 5,466,823 issued to Talley et al.,
many of the celecoxib derivatives described herein have not
previously been prepared. Accordingly, another aspect of the
invention claims the celecoxib derivatives that do not include
benzenesulfonamide, as encompassed by Formula I:
##STR00005##
wherein R.sup.1 is selected from the group consisting of hydrogen,
amino, amido, methylsulfinyl, and ethylsulfinyl moieties, and
R.sup.2 is an aryl group, and wherein R.sup.2 is optionally
substituted at substitutable positions with one of more moieties
independently selected from halo, lower alkyl, lower haloalkyl,
lower hydroxyalkyl, hydroxyl, nitro, amino, carboxyl, and
cyano.
[0039] Further embodiments are directed to these compounds wherein
the R.sup.2 aryl group is selected from the group consisting of
phenyl, naphthyl, biphenyl, anthracenyl, and phenanthracenyl aryl
groups, and further wherein the optional substituting moieties for
R.sup.2 are independently selected from bromo, chloro, fluoro,
methoxy, trifluoromethyl, methyl, ethyl, propyl, and butyl.
[0040] Additional celecoxib compounds that do not include the
benzenesulfaonamide group are
4-(5-biphenyl-4-yl-3-trifluoromethyl-pyrazol-1-yl)-benzamide
(Compound 1);
4-[5-(4'-chloro-biphenyl-4-yl)-3-trifluoromethyl-pyrazol-1-yl]-benzam-
ide (Compound 2);
4-[5-(3',5'-dichloro-biphenyl-4-yl)-3-trifluoromethyl-pyrazol-1-yl]-benza-
mide (Compound 3);
4-[5-(4'-methyl-biphenyl-4-yl)-3-trifluoromethyl-pyrazol-1-yl]-benzamide
(Compound 4);
4-[3-trifluoromethyl-5-(4'-trifluoromethyl-biphenyl-4-yl)-pyrazol-1-yl]-b-
enzamide (Compound 5); 4-[5-(3',5
dimethyl-biphenyl-4-yl)-3-trifluoromethyl-pyrazol-1-yl]-benzamide
(Compound 6);
4-[5-(4'-tert-butyl-biphenyl-4-yl)-3-trifluoromethyl-pyrazol-1-yl]-benzam-
ide (Compound 7);
4-[5-(4'-butyl-biphenyl-4-yl)-3-trifluoromethyl-pyrazol-1-yl]-benzamide
(Compound 8);
4-(5-anthracen-9-yl-3-trifluoromethyl-pyrazol-1-yl)-benzamide
(Compound 9); and
4-[5-(4-butyl-phenyl)-3-trifluoromethyl-pyrazol-1-yl]-benzamide
(Compound 10);
5-(4-fluoro-phenyl)-1-(4-ethanesulfonyl-phenyl)-3-trifluoromethyl-1H-pyra-
zole (Compound 14);
1-(4-methanesulfonyl-phenyl)-5-(4'-methyl-biphenyl-4-yl)-3-trifluoromethy-
l-1H-pyrazole (Compound 15);
4-(5-biphenyl-4-yl-3-trifluoromethyl-pyrazol-1-yl)-phenylamine
(Compound 16);
4-[5-(3',4',5-trichloro-biphenyl-4-yl)-3-trifluoromethyl-pyrazol-1-y-
l]-phenylamine (Compound 17);
4-(5-naphthalen-1-yl-3-trifluoromethyl-pyrazol-1-yl)-phenylamine
(Compound 18);
4-[5-(6-methoxy-naphthalen-2-yl)-3-trifluoromethyl-pyrazol-1-yl]-phenylam-
ine (Compound 19);
4-[5-(4'-bromo-biphenyl-4-yl)-3-trifluoromethyl-pyrazolyl]-phenylamine
(Compound 20); and
5-phenanthren-3-yl-1-phenyl-3-trifluoromethyl-1H-pyrazole (Compound
21), as well as the pharmaceutically acceptable salts of these
compounds.
[0041] Additional embodiments of the invention are directed to
celecoxib derivatives that do not include benzenesulfonamide and
that exhibit relatively high activity. For example, one embodiment
includes the compounds
4-[5-(4'-chloro-biphenyl-4-yl)-3-trifluoromethyl-pyrazol-1-yl]--
benzamide (Compound 2);
[0042] 4-(5-biphenyl-4-yl
3-trifluoromethyl-pyrazol-1-yl)-phenylamine (Compound 16); and
4-[5-(4'-bromo-biphenyl-4-yl)-3-trifluoromethyl-pyrazolyl]-phenylamine
(Compound 20); while a further embodiment is directed specifically
to
4-[5-(4'-bromo-biphenyl-4-yl)-3-trifluoromethyl-pyrazolyl]-phenylamine
(Compound 20).
[0043] Additional embodiments include the pharmaceutically
acceptable salts of these compounds.
Treatment of Francesella tularensis Using Celecoxib Derivatives
[0044] The present invention provides methods for treating or
preventing infection by Francisella tularensis in a subject using
celecoxib derivatives. Francisella tularensis is a pathogenic
species of gram-negative bacteria that is the causative agent of
tularemia; also known as rabbit fever. Francisella tularensis
includes the subspecies tularensis (type A), palearctica (type B),
novicida, and mediasiatica.
[0045] Treatment, as used herein, encompasses both prophylactic and
therapeutic treatment. Celecoxib derivatives of the invention can,
for example, be administered prophylactically to a mammal prior to
exposure to infection by Francisella tularensis. Prophylactic
administration, also referred to as prevention, is effective to
decrease the likelihood of the subsequent infection in the mammal,
or decrease the severity of Francisella infection that subsequently
occurs. Alternatively, celecoxib derivatives of the invention can,
for example, be administered therapeutically to a subject that is
already infected by Francisella tularensis. In one embodiment of
therapeutic administration, administration of the celecoxib
derivatives are effective to eliminate the infection; in another
embodiment, administration of the celecoxib derivatives is
effective to decrease the severity of the infection. The subject is
preferably a mammal, such as a domesticated farm animal (e.g., cow,
horse, pig) or pet (e.g., dog, cat). More preferably, the subject
is a human.
[0046] In some embodiments, the Francisella tularensis infection is
inhibited in macrophage cells, which are the primary in vivo target
for F. tularensis. As shown in the examples provided herein,
celecoxib derivatives are able to effectively inhibit F. tularensis
in macrophages. In additional embodiments, the celecoxib
derivatives are able to inhibit F. tularensis in macrophages
without significant toxicity to other cells, and macrophage cells
in particular.
[0047] The celecoxib derivatives can also be administered to
subjects to treat or prevent infection by antibiotic resistant
strains of Francisella tularensis. Celecoxib has been shown
inhibitory activity against a number of mammalian enzymes,
including phosphoinositide-dependent kinase-1, carbonic anhydrase,
sarcoplasmic/ER calcium ATPase, and COX-1, as further described
herein. Antibiotic resistance can develop through various
mechanisms, such as drug inactivation or modification, alteration
of target site, or alteration of a metabolic pathway that the
antibiotic affects. Accordingly, providing treatment with compounds
having a different structure and different target sites, as
exhibited by the celecoxib derivatives described herein, can
circumvent existing antibiotic resistance in many situations.
Administration and Formulation of Celecoxib Derivatives
[0048] The present invention also provides pharmaceutical
compositions that include celecoxib derivatives according to
formula I as an active ingredient, and a pharmaceutically
acceptable liquid or solid carrier or carriers, in combination with
the active ingredient. Any of the compounds described above as
being suitable for the treatment of Francisella tularensis can be
included in pharmaceutical compositions of the invention.
[0049] The celecoxib derivatives can be administered as
pharmaceutically acceptable salts. Pharmaceutically acceptable salt
refers to the relatively non-toxic, inorganic and organic acid
addition salts of the celecoxib derivatives. These salts can be
prepared in situ during the final isolation and purification of the
celecoxib derivative, or by separately reacting a purified
celecoxib derivative with a suitable counterion, depending on the
nature of the celecoxib derivative, and isolating the salt thus
formed. Representative counterions include the chloride, bromide,
nitrate, ammonium, sulfate, tosylate, phosphate, tartrate,
ethylenediamine, and maleate salts, and the like. See for example
Haynes et al., J. Pharm. Sci., 94, p. 2111-2120 (2005).
[0050] The pharmaceutical compositions includes one or more
celecoxib derivatives together with one or more of a variety of
physiological acceptable carriers for delivery to a patient,
including a variety of diluents or excipients known to those of
ordinary skill in the art. For example, for parenteral
administration, isotonic saline is preferred. For topical
administration, a cream, including a carrier such as
dimethylsulfoxide (DMSO), or other agents typically found in
topical creams that do not block or inhibit activity of the
peptide, can be used. Other suitable carriers include, but are not
limited to, alcohol, phosphate buffered saline, and other balanced
salt solutions.
[0051] The formulations may be conveniently presented in unit
dosage form and may be prepared by any of the methods well known in
the art of pharmacy. Preferably, such methods include the step of
bringing the active agent into association with a carrier that
constitutes one or more accessory ingredients. In general, the
formulations are prepared by uniformly and intimately bringing the
active agent into association with a liquid carrier, a finely
divided solid carrier, or both, and then, if necessary, shaping the
product into the desired formulations. The methods of the invention
include administering to a subject, preferably a mammal, and more
preferably a human, the composition of the invention in an amount
effective to produce the desired effect. The celecoxib derivatives
can be administered as a single dose or in multiple doses. Useful
dosages of the active agents can be determined by comparing their
in vitro activity and the in vivo activity in animal models.
Methods for extrapolation of effective dosages in mice, and other
animals, to humans are known in the art; for example, see U.S. Pat.
No. 4,938,949.
[0052] The agents of the present invention are preferably
formulated in pharmaceutical compositions and then, in accordance
with the methods of the invention, administered to a subject, such
as a human patient, in a variety of forms adapted to the chosen
route of administration. The formulations include, but are not
limited to, those suitable for oral, rectal, vaginal, topical,
nasal, ophthalmic, or parental (including subcutaneous,
intramuscular, intraperitoneal, intratumoral, and intravenous)
administration.
[0053] Formulations of the present invention suitable for oral
administration may be presented as discrete units such as tablets,
troches, capsules, lozenges, wafers, or cachets, each containing a
predetermined amount of the active agent as a powder or granules,
as liposomes containing the celecoxib derivatives, or as a solution
or suspension in an aqueous liquor or non-aqueous liquid such as a
syrup, an elixir, an emulsion, or a draught. Such compositions and
preparations typically contain at least about 0.1 wt-% of the
active agent. The amount of celecoxib derivatives (i.e., active
agent) is such that the dosage level will be effective to produce
the desired result in the subject.
[0054] Nasal spray formulations include purified aqueous solutions
of the active agent with preservative agents and isotonic agents.
Such formulations are preferably adjusted to a pH and isotonic
state compatible with the nasal mucous membranes. Formulations for
rectal or vaginal administration may be presented as a suppository
with a suitable carrier such as cocoa butter, or hydrogenated fats
or hydrogenated fatty carboxylic acids. Ophthalmic formulations are
prepared by a similar method to the nasal spray, except that the pH
and isotonic factors are preferably adjusted to match that of the
eye. Topical formulations include the active agent dissolved or
suspended in one or more media such as mineral oil, petroleum,
polyhydroxy alcohols, or other bases used for topical
pharmaceutical formulations.
[0055] The tablets, troches, pills, capsules, and the like may also
contain one or more of the following: a binder such as gum
tragacanth, acacia, corn starch or gelatin; an excipient such as
dicalcium phosphate; a disintegrating agent such as corn starch,
potato starch, alginic acid, and the like; a lubricant such as
magnesium stearate; a sweetening agent such as sucrose, fructose,
lactose, or aspartame; and a natural or artificial flavoring agent.
When the unit dosage form is a capsule, it may further contain a
liquid carrier, such as a vegetable oil or a polyethylene glycol.
Various other materials may be present as coatings or to otherwise
modify the physical form of the solid unit dosage form. For
instance, tablets, pills, or capsules may be coated with gelatin,
wax, shellac, sugar, and the like. A syrup or elixir may contain
one or more of a sweetening agent, a preservative such as methyl-
or propylparaben, an agent to retard crystallization of the sugar,
an agent to increase the solubility of any other ingredient, such
as a polyhydric alcohol, for example glycerol or sorbitol, a dye,
and flavoring agent. The material used in preparing any unit dosage
form is substantially nontoxic in the amounts employed. The active
agent may be incorporated into sustained-release preparations and
devices.
Preparation of the Compounds
[0056] Compounds of the invention may be synthesized by synthetic
routes that include processes similar to those well known in the
chemical arts, particularly in light of the description contained
herein. The starting materials are generally available from
commercial sources such as Aldrich Chemicals (Milwaukee, Wis., USA)
or are readily prepared using methods well known to those skilled
in the art (e.g., prepared by methods generally described in Louis
F. Fieser and Mary Fieser, Reagents for Organic Synthesis, v. 1-19,
Wiley, New York, (1967-1999 ed.); Alan R. Katritsky, Otto
Meth-Cohn, Charles W. Rees, Comprehensive Organic Functional Group
Transformations, v 1-6, Pergamon Press, Oxford, England, (1995);
Barry M. Trost and Ian Fleming, Comprehensive Organic Synthesis, v.
1-8, Pergamon Press, Oxford, England, (1991); or Beilsteins
Handbuch der organischen Chemie, 4, Aufl. Ed. Springer-Verlag,
Berlin, Germany, including supplements (also available via the
Beilstein online database)).
[0057] Those skilled in the art will appreciate that other
synthetic routes may be used to synthesize the compounds of the
invention. Although specific starting materials and reagents are
depicted in the reaction schemes and discussed below, other
starting materials and reagents can be easily substituted to
provide a variety of derivatives and/or reaction conditions. In
addition, many of the compounds prepared by the methods described
below can be further modified in light of this disclosure using
conventional methods well known to those skilled in the art.
[0058] The present invention is illustrated by the following
examples. It is to be understood that the particular examples,
materials, amounts, and procedures are to be interpreted broadly in
accordance with the scope and spirit of the invention as set forth
herein.
EXAMPLES
Example 1
General Procedures for 1,1,1-trifluoro-4-aryl-but-3-en-2-one
Intermediates
##STR00006##
[0060] To a suspension of sodium hydride (NaH; 0.13 g, 5.4 mmol) in
5 ml of anhydrous tetrahydrofuran (THF) was added ethyl
trifluoroacetate (CF.sub.3COOEt; 0.64 g, 4.5 mmol) under argon.
After stirring at 25.degree. C. for 10 min, 4-substituted phenyl
(4.5 mmol) in 5 ml of THF was added dropwise to the solution. The
mixture became clear and orange hued within 30 min, and after
stirring for an additional 2 h, was concentrated under vacuum. The
residue was suspended in water, and extracted with ethyl acetate
(15 ml) twice. The organic phase was separated, dried over sodium
sulfate, and concentrated to dryness under vacuum to give the
product (yellow solid; 1.29 g, 90% yield). The product was used
directly without purification. [0061]
1,1,1-Trifluoro-4-hydroxy-4-(4'-methyl-biphenyl-4-yl)-but-3-en-2-one;
[0062]
4-(4'-Chloro-biphenyl-4-yl)-1,1,1-trifluoro-4-hydroxy-but-3-en-2-o-
ne; [0063] 4-(3',
5'-Dichloro-biphenyl-4-yl)-1,1,1-trifluoro-4-hydroxy-but-3-en-2-one;
[0064]
4-(4'-Methyl-biphenyl-4-yl)-1,1,1-trifluoro-4-hydroxy-but-3-en-2-o-
ne; [0065]
4-(4'-Trifluoromethyl-biphenyl-4-yl)-1,1,1-trifluoro-4-hydroxy--
but-3-en-2-one; [0066] 4-(3',
5'-Dimethyl-biphenyl-4-yl)-1,1,1-trifluoro-4-hydroxy-but-3-en-2-one;
[0067]
4-(4'-t-Butyl-biphenyl-4-yl)-1,1,1-trifluoro-4-hydroxy-but-3-en-2--
one; [0068]
4-(4'-n-Butyl-biphenyl-4-yl)-1,1,1-trifluoro-4-hydroxy-but-3-en-2-one;
[0069] 4-Anthracen-9-yl-1,1,1-trifluoro-4-hydroxy-but-3-en-2-one;
[0070] 4-(4-Butyl-phenyl)-1,1,1-trifluoro-4-hydroxy-but-3-en-2-one;
[0071] 4-(2',4',
5'-Trichloro-biphenyl-4-yl)-1,1,1-trifluoro-4-hydroxy-but-3-en--
2-one; [0072]
1,1,1-Trifluoro-4-hydroxy-4-naphthalen-1-yl-but-3-en-2-one; [0073]
4-(4-Fluoro-phenyl)-1,1,1-trifluoro-4-hydroxy-but-3-en-2-one;
[0074]
1,1,1-Trifluoro-4-hydroxy-4-(6-methoxy-naphthalen-2-yl)-but-3-en-2-
-one; [0075]
4-(4'-Bromo-biphenyl-4-yl)-1,1,1-trifluoro-4-hydroxy-but-3-en-2-one;
[0076]
1,1,1-Trifluoro-4-hydroxy-4-phenanthren-3-yl-but-3-en-2-one.
Example 2
General Procedures for 4-Carboxamide Compounds
##STR00007##
[0078] (4-Carbamoylphenyl)-hydrazine hydrochloride (0.92 g, 4.9
mmol) was added to a stirred solution of precursors (I to X) (4.1
mmol) in 40 ml of ethanol at 25.degree. C. to prepare compounds
1-10, below. The mixture was refluxed for 12 h, cooled to room
temperature and concentrated to dryness under vacuum. The residue
was dissolved in ethyl acetate, and washed with water. The organic
layer was dried over sodium sulfate, and concentrated under vacuum.
The crude product was purified by silica gel flash chromatography
(ethyl acetate-hexane, 1:1), yielding 1 to 10 respectively in good
yield. [0079] Compound 1:
4-(5-Biphenyl-4-yl-3-trifluoromethyl-pyrazol-1-yl)-benzamide;
[0080] Compound 2:
4-[5-(4'-Chloro-biphenyl-4-yl)-3-trifluoromethyl-pyrazol-1-yl]-benzamide;
[0081] Compound 3: 4-[5-(3',
5'-Dichloro-biphenyl-4-yl)-3-trifluoromethyl-pyrazol-1-yl]-benzamide;
[0082] Compound 4:
4-[5-(4'-Methyl-biphenyl-4-yl)-3-trifluoromethyl-pyrazol-1-yl]-benzamide;
[0083] Compound 5:
4-[3-Trifluoromethyl-5-(4'-trifluoromethyl-biphenyl-4-yl)-pyrazol-1-yl]-b-
enzamide; [0084] Compound 6:
4-[5-(3,5'-Dimethyl-biphenyl-4-yl)-3-trifluoromethyl-pyrazol-1-yl]-benzam-
ide; [0085] Compound 7:
4-[5-(4'-t-Butyl-biphenyl-4-yl)-3-trifluoromethyl-pyrazol-1-yl]-benzamide-
; [0086] Compound 8:
4-[5-(4'-n-Butyl-biphenyl-4-yl)-3-trifluoromethyl-pyrazol-1-yl]-benzamide-
; [0087] Compound 9:
4-(5-Anthracen-9-yl-3-trifluoromethyl-pyrazol-1-yl)-benzamide;
[0088] Compound 10:
4-[5-(4-Butyl-phenyl)-3-trifluoromethyl-pyrazol-1-yl]-benzamide.
Example 3
General Procedures for 4-Sulfonamide Compounds (11-13)
##STR00008##
[0090] 4-Hydrazinobenzene-1-sulfonamide hydrochloride (1.1 g, 4.9
mmol) was added to a stirred solution of precursors (II, XI and
XII) (4.1 mmol) in 40 ml of ethanol. The mixture was refluxed for
12 h, cooled to room temperature, and concentrated to dryness under
vacuum. The residue was dissolved in ethyl acetate, and washed with
water. The organic layer was dried over sodium sulfate, and
concentrated under vacuum. The crude product was purified by silica
gel flash chromatography to yield 11, 12, 13 respectively. [0091]
Compound 11:
4-[5-(4'-Chloro-biphenyl-4-yl)-3-trifluoromethyl-pyrazol-1-yl]-benzenesul-
fonamide; [0092] Compound 12:
4-[5-(2',4',5'-Trichloro-biphenyl-4-yl)-3-trifluoromethyl-pyrazol-1-yl]-b-
enzenesulfonamide; [0093] Compound 13:
4-(5-Naphthalen-1-yl-3-trifluoromethyl-pyrazol-1-yl)-benzenesulfonamide.
Example 4
General Procedures for 4-Methanesulfonyl Compounds
##STR00009##
[0095] (4-Methanesulfonyl-phenyl)-hydrazine hydrochloride (4.9
mmol) was added to a stirred solution of precursors (XIII, IV) (4.1
mmol) in 40 ml of ethanol. The mixture was refluxed for 12 h,
cooled to room temperature, and concentrated to dryness under
vacuum. The residue was dissolved in ethyl acetate, and washed with
water. The organic layer was dried over sodium sulfate, and
concentrated under vacuum. The crude product was purified by silica
gel flash chromatography to yield 14 and 15 respectively. [0096]
Compound 14:
5-(4-Fluoro-phenyl)-1-(4-methanesulfonyl-phenyl)-3-trifluoromethyl-1H-pyr-
azole; [0097] Compound 15:
1-(4-Methanesulfonyl-phenyl)-5-(4'-methyl-biphenyl-4-yl)-3-trifluoromethy-
l-1H-pyrazole.
Example 5
General Procedures for 4-Amine Compounds
##STR00010##
[0099] To a solution of precursors I, XI, XII, XIV, and XV (4.1
mmol) in 40 ml of ethanol was added 4-nitrophenylhydrazine
hydrochloride (4.9 mmol) respectively under stirring, refluxed for
1 h, cooled to room temperature, and concentrated to dryness under
vacuum. The residue was dissolved in ethyl acetate, and washed with
water. The organic phase was dried over magnesium sulfate, and
concentrated to dryness under vacuum. The crude product was
purified by silica gel column chromatography to afford nitro
compounds XVII, XVIII, XIX, XX, and XXI respectively in fair to
good yield. [0100] XVII.
5-Biphenyl-4-yl-1-(4-nitro-phenyl)-3-trifluoromethyl-1H-pyrazole
[0101] XVIII.
5-(2',4',5'-Trichloro-biphenyl-4-yl)-1-(4-nitro-phenyl)-3-trifluor-
omethyl-1H-pyrazole [0102] XIX.
5-Naphthalen-1-yl-1-(4-nitro-phenyl)-3-trifluoromethyl-1H-pyrazole
[0103] XX.
5-(6-methoxy-naphthalen-2-yl)-1-(4-nitro-phenyl)-3-trifluoromethyl-1H-
-pyrazole [0104] XXI.
5-(4'-Bromo-biphenyl-4-yl)-1-(4-nitro-phenyl)-3-trifluoromethyl-1H-pyrazo-
le
[0105] To a solution of nitro compounds XVII, XVIII, XIX, XX, and
XXI (2 mmol) in 20 ml ethanol was added platinum oxide (27 mg, 0.12
mmol) respectively, stirred under H.sub.2 at 55 psi for 12 h,
filtered to remove the catalyst, and concentrated to dryness under
vacuum. The crude product was purified by silica gel column
chromatography to yield compounds 16, 17, 18, 19, and 20
respectively in good yield. [0106] Compound 16:
4-(5-Biphenyl-4-yl-3-trifluoromethyl-pyrazol-1-yl)-phenylamine;
[0107] Compound 17:
4-[5-(2',4',5'-Trichloro-biphenyl-4-yl)-3-trifluoromethyl-pyrazol-1-yl]-p-
henylamine; [0108] Compound 18:
4-(5-Naphthalen-1-yl-3-trifluoromethyl-pyrazol-1-yl)-phenylamine;
[0109] Compound 19:
4-[5-(6-methoxy-naphthalen-2-yl)-3-trifluoromethyl-pyrazol-1-yl)-phenylam-
ine; [0110] Compound 20:
4-[5-(4'-Bromo-biphenyl-4-yl)-3-trifluoromethyl-pyrazol-1-yl]-phenylamine-
.
Example 6
1-Phenyl-5-phenanthrenyl-3-(trifluoromethyl)-1H-pyrazole (21)
##STR00011##
[0112] To a solution of XVI (4.1 mmol) in 40 ml of ethanol was
added phenylhydrazine hydrochloride (4.9 mmol) under stirring,
refluxed for 1 h, cooled to room temperature, and concentrated to
dryness under vacuum. The residue was dissolved in ethyl acetate,
and washed with water. The organic phase was dried over magnesium
sulfate, and concentrated to dryness under vacuum. The crude
product was purified by silica gel column chromatography to afford
compound 21,
(5-phenanthren-3-yl-1-phenyl-3-trifluoromethyl-1H-pyrazole) (0.88
g, 50% yield).
Example 7
Antibacterial Effect of Celecoxib and Celecoxib Derivatives Against
Francisella tularensis
Materials and Methods
Bacteria
[0113] F. novicida strain U112, the live vaccine strain (LVS, type
B), and F. tularensis strain Schu S4 (type A) were used throughout
this study. Experiments involving Schu S4 were conducted in a CDC
select agent-approved BSL3 laboratory at Ohio State University.
Bacteria were grown at 37.degree. C. on chocolate II agar (Becton,
Dickinson and Company, Franklin Lakes, N.J.) or in tryptic soy
broth (Becton, Dickinson and Company) supplemented with 0.025%
(w/v) iron (III) pyrophosphate (Sigma-Aldrich, St. Louis, Mo.) and
0.1% (w/v) cysteine hydrochloride (MP Biomedicals, Solon,
Ohio).
[0114] Salmonella enterica serovar Typhimurium (ATCC 14028) and
Escherichia coli (ATCC 25922) were grown on Luria-Bertani (LB) agar
(Becton, Dickinson and Company) or in LB broth at 37.degree. C.
Experiments involving these bacteria were performed using biosafety
level 2 (BSL-2) laboratory procedures.
Reagents
[0115] Celecoxib was extracted and purified from Celebrex capsules
(Amerisource Health, Malvern, Pa.) with ethyl acetate followed by
recrystallization in a mixture of ethyl acetate and hexane.
Rofecoxib was synthesized according the procedure of Prasit et al.
Prasit et al., Bioorg. Med. Chem. Lett. 9, p. 1773-8 (1999). The
celecoxib-based compound library consisted of compounds 1-21, as
described in Examples 2-6. The identity and purity (>99%) of
these synthetic compounds were verified by proton nuclear magnetic
resonance spectroscopy (300 MHz), high resolution mass
spectrometry, and elemental analysis.
Macrophages
[0116] The RAW264.7 murine macrophage cell line and THP-1 human
monocytic leukemia cell line were obtained from American Type
Culture Collection (ATCC; Manassas, Va.). The RAW264.7 cells were
maintained in Dulbecco's Modified Eagle's Medium (DMEM) (GIBCO-BRL,
Invitrogen Corp., Carlsbad, Calif.) supplemented with 10% FBS
(GIBCO-BRL). The THP-1 cells were maintained in RPMI 1640
containing 10% FBS. THP-1 cells were differentiated by treatment
with 20 nM of 12-O-tetradecanoylphorbol 13-acetate (TPA)
(Sigma-Aldrich, St. Louis, Mo.) for 48 h. All culture of murine and
human cells was performed at 37.degree. C. in a humidified
incubator containing 5% CO2. Cells were seeded into 96- or 12-well
tissue culture plates, and incubated for 8-12 hours prior to
experimentation.
Antibacterial Assays
[0117] The minimum inhibitory concentration (MIC) of individual
agents was determined by a broth microdilution method as described
below. MIC was defined as the lowest concentration that
significantly inhibited the bacterial growth. F. tularensis cells
grown overnight on a chocolate II agar plate were suspended in PBS
to an O.D. of 1.0 at 600 nm, which was equivalent to 10.sup.10
CFU/ml, and then diluted in modified TSB to a final concentration
of 10.sup.4 CFU/ml. The bacterial suspension was exposed to the
test agent at escalating doses, ranging from 1-64 .mu.g/ml, in
triplicate in 96-well plates, and incubated at 37.degree. C. for 24
h (F. novicida and Schu S4) or 48 h (LVS). The MIC was derived from
the concentration that exhibited no visible growth of bacteria.
Subsequently, to analyze the viability of F. novicida and LVS after
drug exposure, 100 .mu.l of bacterial suspension from each well was
serially diluted with PBS and spread onto chocolate II agar plates.
After 24 h (F. novicida) or 48 h (LVS) of incubation at 37.degree.
C., the number of bacterial colonies on each plate was counted and
expressed as CFU/ml. The effects of test agents on the growth of
two additional Gram-negative bacteria, S. enterica serovar
Typhimurium and E. coli, in LB broth or modified TSB were assayed
as described above for Francisella spp.
Macrophage Viability Assay
[0118] The effect of individual agents on macrophage viability was
assessed by using the
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) assay.
Jeffrey, M. E., Methods in Cell Science, 11(1), p. 3 (1988).
RAW264.7 cells were seeded into 96-well plates at
2.5.times.10.sup.4 cells/well (minimum of six wells per test group)
in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10%
fetal bovine serum (FBS) and 10 .mu.g/ml of gentamicin, and then
incubated overnight at 37.degree. C. in a humidified incubator
containing 5% CO.sub.2. The medium from each well was removed and
replaced with fresh 5% FBS-supplemented DMEM containing various
concentrations of test agents dissolved in DMSO (final
concentration of 0.1%). Controls received DMSO alone at a
concentration equal to that in drug-treated cells. After 8 h of
treatment, the medium was removed, replaced by 200 .mu.A of 0.5
mg/ml of MTT in 10% FBS-containing medium, and the cells were
incubated in the CO.sub.2 incubator at 37.degree. C. for 1 h.
Supernatants were removed from the wells, and the reduced MTT dye
was solubilized in 200 .mu.l/well of DMSO. Absorbances at 570 nm
were determined on a plate reader. The viability of drug-treated
cells was calculated as a percentage of vehicle-treated control
cells, and an IC.sub.50 for cell viability was determined by using
CalcuSyn software (Biosoft, Cambridge, UK).
Assay for Intracellular Survival of Francisella in Macrophages
[0119] F. novicida cells grown overnight on a chocolate II agar
plate were suspended in PBS to a concentration of approximately
10.sup.10 CFU/ml (as estimated by an O.D. of 1.0 at 600 nm).
RAW264.7 murine macrophages and TPA-differentiated THP-1 cells were
seeded into 12-well plates at 5.times.10.sup.5 cells/well, and F.
novicida was added at an MOI of 50. Mohaptra et al., Infect. Immun.
76, p. 3690-9 (2008). After centrifugation of plates at 800.times.g
for 15 min to facilitate infection, macrophages were incubated at
37.degree. C. in a humidified incubator containing 5% CO.sub.2 for
2 h, exposed to 50 .mu.g/ml of gentamicin for 30 min, and washed
with pre-warmed PBS twice to remove killed extracellular bacteria.
Mariathasan et al., J. Exp. Med. 202, p. 1043-9 (2005). Infected
macrophages were then treated in triplicate with various
concentrations of test agents for 8 h, after which culture medium
was collected from each well and macrophages lysed with 500 .mu.l
of 0.1% sodium deoxycholate in PBS at 37.degree. C. for 5 min to
release intracellular bacteria. Mohaptra et al., Infect. Immun.,
75, p. 390-6 (2007). Bacteria present in the collected culture
medium, either as free bacteria or within floating macrophages,
were harvested by centrifugation at 16,000.times.g for 5 min,
followed by resuspension of the pellet in 500 .mu.l of 0.1% sodium
deoxycholate in PBS. Combined lysates were serially diluted with
PBS and spread onto chocolate II agar plates. CFU were calculated
after incubation for 24 h at 37.degree. C. Survival of
intracellular bacteria in drug-treated macrophages was calculated
as a percentage of control (untreated) cells.
Statistical Analysis
[0120] Data are expressed as means.+-.SD. Group means were compared
using a two-tailed t-test for independent samples. Differences were
considered significant at P<0.05. Statistical analyses were
performed using SPSS for Windows (Version 16.0; SPSS, Inc. Chicago,
Ill.).
Results
Differential Effect of Celecoxib and Rofecoxib on Inhibiting the
Growth of F. Tularensis in Broth Culture.
[0121] As part of the inventors' effort to identify lead agents
with antibacterial activity against F. tularensis, the effect of a
panel of pharmaceuticals in clinical use on the growth of F.
novicida and LVS in modified tryptic soy broth (TSB) was examined.
Of the drugs examined, the cyclooxygenase-2 (COX-2) inhibitor
celecoxib exhibited a significant ability to inhibit bacterial
growth with MIC values of 32 .mu.g/ml and 16 .mu.g/ml for F.
novicida and LVS, respectively (FIG. 1). As shown, treatment of
these bacteria with celecoxib at the respective MICs led to a
.gtoreq.log decrease in CFU, and bacteria were completely
eliminated at 64 .mu.g/ml and 32 .mu.g/ml, respectively (FIG. 1B).
Importantly, celecoxib was equipotent in suppressing the growth of
the human virulent type A strain of F. tularensis Schu S4 with an
MIC of 16 .mu.g/ml (FIG. 1C). Moreover, this suppressive effect was
highly specific against Francisella since celecoxib was inactive
against two other gram-negative bacteria examined, namely S.
enterica serovar Typhimurium and E. coli. In contrast, rofecoxib, a
structurally distinct but more potent COX-2 inhibitor, had no
appreciable effect on any of the bacteria examined at 64 .mu.g/ml
(FIGS. 1B and C), indicating that the antibacterial effect of
celecoxib was attributable to an "off-target" mechanism independent
of inhibiting a putative "COX-2-like" enzyme in Francisella.
Pharmacological Exploitation of the Anti-Francisella Activity of
Celecoxib.
[0122] It was determined that the findings of an "off-target"
antibacterial activity of celecoxib against F. tularensis could be
exploited by using celecoxib as a molecular platform to develop
additional potent anti-Francisella agents for therapeutic use.
Accordingly, a focused compound library consisting of twenty-one
celecoxib derivatives was established by replacing the methylphenyl
(R.sub.1) and sulfonamide (R.sub.2) fragments of celecoxib with
various functionalities. The MICs of these compounds for bacterial
growth inhibition were measured following growth in modified TSB
after 24 h (for F. novicida) or 48 h (for LVS) at 37.degree. C., as
shown in FIG. 2. Of them, compounds 2, 11, 12, 16, and 20 exhibited
MICs of no greater than 4 .mu.g/ml for both strains. In particular,
compound 12 was able to suppress the growth of F. novicida and LVS
at 2 and 1 .mu.g/ml, respectively. This multifold increase in
antibacterial activity showed that celecoxib could be structurally
optimized to develop potent anti-Francisella agents.
Differential Cytotoxicity of Lead Agents for Macrophages.
[0123] Since the primary in vivo target for F. tularensis is the
macrophage, the cytotoxicity of celecoxib and these lead agents was
further assessed in RAW264.7 murine macrophage cells. FIG. 3A
depicts the dose-response effects of individual agents on the death
of RAW264.7 cells in 5% FBS-containing DMEM medium after 8-h
treatment, of which the relative potency is in the order of
12>11>2>16>celecoxib>20. Serum was an important
variable in this assay as prior studies had shown that serum can
suppress the activity of these agents.
[0124] Although compound 12 at 2 .mu.g/ml was highly effective in
inhibiting bacterial growth, it also showed cytotoxicity for
macrophages at the same concentration, i.e., IC.sub.50/MIC ratio of
1.2 (FIG. 3B). On the other hand, compound 20 exhibited the highest
IC.sub.50/MIC ratio of 11.5, indicating a desirable selectivity in
drug-induced bacterial growth inhibition relative to cytotoxicity
for host cells. Moreover, like celecoxib, the inhibitory activity
of compound 20 was specific for Francisella as it was inactive
against the gram-negative bacteria S. enterica serovar Typhimurium
and E. coli (data not shown).
[0125] The 50% inhibitory concentration (IC.sub.50) of each agent
tested from three independent experiments was calculated by using
CalcuSyn software. The ratio of the MIC (bacteria growth
inhibition) to the IC.sub.50 (cell toxicity) for each agent was
also calculated and served as an index of selectivity for
antimicrobial activity relative to a cytotoxic effect (the higher
the number the greater the selectivity).
[0126] Compound 20 inhibits the growth of intracellular F.
tularensis in murine and human macrophages. Based on these results,
compound 20 was further studied for its effect on inhibiting the
intracellular survival of F. novicida in murine RAW264.7 and
12-O-tetradecanoyl-phorbol-13-acetate (TPA)-differentiated human
THP-1 macrophages. After infection and removal of extracellular
bacteria, infected macrophages were treated with 4, 8 and 16 ng/ml
of compound 20 in 5% FBS-containing DMEM medium for 8 h.
Intracellular bacteria were then harvested and enumerated by
calculating CFU after growth on agar. As shown in FIG. 4A, compound
20 effectively inhibits intracellular F. novicida at 16 .mu.g/ml
(P<0.05). Subsequently, the effect of compound 20 on
intracellular F. tularensis (type A, Schu S4) in TPA-treated THP-1
cells was assayed. At 4 .mu.g/ml, compound 20 already showed
significant inhibitory effect on the intracellular Schu S4
(P<0.01), indicating the higher susceptibility of Schu S4 to
compound 20 than F. novicida (FIG. 4B).
Discussion
[0127] Several drugs that originally were not developed for the
treatment of bacterial infections have been demonstrated to possess
antimicrobial activity in vitro. For example, the
cholesterol-lowering drug, statin, was shown to inhibit the in
vitro growth of Staphylococcus aureus. Jerwood et al., J.
Antimicrob. Chemother. 61, p. 362-4 (2008). The results provided
herein demonstrated that celecoxib, a broadly used
anti-inflammatory agent, exhibits an "off-target" antibacterial
activity against F. tularensis in vitro. It is particularly
noteworthy that the antimicrobial activity of celecoxib against F.
tularensis is more potent than against F. novicida. This
differential effect is reflected in the disparate MICs for F.
novicida versus F. tularensis (type A Schu S4) and LVS. Moreover,
the assessment of the anti-Francisella activity of new celecoxib
derivatives revealed that F. novicida and LVS show a marked
difference in their susceptibilities to celecoxib and its
derivatives, especially compound 17, which had no measurable
inhibitory effect on F. novicida, but was a potent inhibitor of LVS
growth in modified TSB (FIG. 2). This finding indicates that the
interaction between the drug and its putative bacterial target
protein differs between Francisella spp. A possibility is that the
binding site for celecoxib on its putative target protein differs,
leading to higher binding affinity in F. tularensis strains, and
stronger growth inhibition in vitro.
[0128] Among the celecoxib derivatives synthesized and evaluated,
compound 20 was identified as having the best selectivity for its
bacterial growth inhibitory effects relative to its cytotoxicity
for macrophages. Equally important, it could inhibit the survival
of intracellular Francisella in both murine and human macrophages
although at a higher concentration than the MIC for bacteria grown
in broth culture (16 .mu.g/ml versus 4 .mu.g/ml). This discrepancy
reflects many factors that limit the access of antibacterial agents
to intracellular pathogens, including serum protein binding and
physical barriers imposed by biological membranes.
[0129] Because Francisella is primarily located in macrophages of
the infected host, methods for targeted drug delivery to
macrophages should be considered in the development of next
generation anti-Francisella agents. For instance, strategies that
couple compound 20 with a carrier that can be actively phagocytosed
by macrophages may prove to be a promising means to attain both
increased intracellular drug concentrations and specificity of drug
delivery. One possible approach in this regard is to utilize the
mannose receptor that is expressed abundantly on macrophages, which
has been broadly used to enhance the specific delivery of drugs,
oligonucleotides and proteins to intracellular compartments in
macrophages. Irache et al., Expert Opin. Drug Deliv., 5, p. 703-24
(2008).
[0130] Celecoxib and rofecoxib are potent COX-2 inhibitors that
have been shown to interact with the same binding pocket of the
COX-2 enzyme with IC.sub.50 values in the sub-.mu.M range.
Nonetheless, the data show that only celecoxib possessed
antimicrobial activity toward Francisella, and that the MIC of
celecoxib for Francisella growth inhibition (32 .mu.g/ml) is much
higher than its reported IC.sub.50 for COX-2 inhibition (0.21
.mu.g/ml) Prasit et al., Bioorg. Med. Chem. Lett., 9, p. 1773-8
(1999). These findings suggest that the antimicrobial activity of
celecoxib is independent of the structural features that dictate
its binding to COX-2. Thus, it appears likely that the putative
bacterial target of celecoxib in F. tularensis is structurally
distinct from the COX-2 enzyme. In addition to COX-2, celecoxib has
been reported to possess inhibitory activity against other
mammalian enzymes, including phosphoinositide-dependent kinase-1,
carbonic anhydrase, sarcoplasmic/ER calcium ATPase (SERCA), and
COX-1. Schonthal, A. H., Br. J. Cancer, 97, p. 1465-8 (2007). These
mammalian enzymes may serve as leads to identify structurally
similar bacterial proteins, of which one could be the putative
antibacterial target of celecoxib in F. tularesis.
[0131] Accordingly, the protein sequences of these
celecoxib-targeted enzymes were used to search for homologous
protein sequences in the published proteome of F. tularensis (Schu
S4), F. novicida and LVS at the National Center of Biotechnology
Information (http://www.ncbi.nlm.nih.gov/). The results identified
several proteins of F. novicida and LVS that share homology with
carbonic anhydrase, SERCA and COX-1, which include superoxide
dismutase, FGAM synthase and a cation transport ATPase (FTF1738c).
These findings suggest that such an approach to identifying
bacterial drug targets is feasible, and will facilitate the
development of more potent and specific, celecoxib-derived
anti-Francisella agents.
[0132] The complete disclosure of all patents, patent applications,
and publications, and electronically available material cited
herein are incorporated by reference. The foregoing detailed
description and examples have been given for clarity of
understanding only. No unnecessary limitations are to be understood
therefrom. The invention is not limited to the exact details shown
and described, for variations obvious to one skilled in the art
will be included within the invention defined by the claims.
[0133] All headings are for the convenience of the reader and
should not be used to limit the meaning of the text that follows
the heading, unless so specified.
* * * * *
References